Aminoglycosides have been proposed as therapies for genetic disorders caused by nonsense mutations, because of their capacity to enhance translational read-through of premature termination codons (PTCs), thereby permitting expression of functional full-length protein. However, a potential consequence of this strategy is the development of an autoimmune response to HLA-presented epitopes encoded downstream of the PTC or other stop codons. Using a recombinant virus-expression system in tissue culture and in mice, we demonstrate that gentamicin can induce expression and MHC class I presentation of a model epitope encoded downstream of a PTC at levels sufficient to activate CD8⁺ T cells. The degree of read-through–derived peptide presentation varies with the sequence of the stop codon and +1 nucleotide. Additionally, we applied a mass spectrometry exploration of the HLA class I peptide repertoire of gentamicin-treated cells and identified multiple peptides derived from read-through of conventional stop codons. These results substantiate the possibility of self-reactivity to cryptic epitopes revealed by stop codon read-through therapies and potentially other therapeutic approaches involving compounds that alter translational fidelity.